卵巢透明细胞癌ES2细胞中ARID1A基因沉默对顺铂敏感性的影响及相关研究

发布时间：2018-05-05 07:49

本文选题：卵巢透明细胞癌 + siRNA　；参考：《北京协和医学院》2016年博士论文

【摘要】：研究目的：体外设计并合成靶向于人ARID1A基因的siRNA,转染人卵巢透明细胞癌ES2细胞,筛选出下调ARID1A表达的有效片段,研究ARID1A基因沉默对顺铂敏感性的影响,探讨可能涉及的分子通路改变；研究蛋白激酶B抑制剂哌立福辛联合顺铂对ES2细胞的体外增殖抑制作用,探讨二者联合的协同作用及可能的促凋亡机制,为今后卵巢透明细胞癌的分子靶向治疗提供一定的理论依据。研究方法：体外合成各组siRNA并瞬时转染ES2细胞；利用RT-PCR法和western blot法分别从mRNA和蛋白水平检测ARID1A基因沉默情况,筛选出最佳片段(即siRNA-3)。使用siRNA-3片段瞬时转染ES2细胞,24 h后加入不同浓度的顺铂,孵育48 h后采用CCK-8法检测各实验组半数抑制浓度(IC50)；转染后24h加入相同浓度的顺铂50 μM,孵育48 h后测得各组细胞生存率；采用流式细胞仪检测各实验组ES2细胞的凋亡率：采用western blot法检测各实验组ES2细胞中蛋白激酶B(AKT)表达水平的变化。以不同浓度哌立福辛作用于ES2细胞48 h,采用CCK-8法检测细胞增殖抑制情况；以哌立福辛最小有作用剂量与不同浓度顺铂联合处理细胞48 h,以金氏公式筛选出协同抑制作用最明显的联合剂量,以该联合剂量处理ES2细胞48 h,采用DAPI染色法及流式细胞仪检测细胞的凋亡情况；采用Giemsa染色法检测细胞有丝分裂指数；采用western blot法检测细胞中Caspase-3(cleaved)及PARP(cleaved)蛋白表达水平的变化。研究结果：使用ARID1A特异性siRNA-3片段转染ES2细胞后,CCK-8法结果显示：siRNA-3组的IC50较siRNA-NC组和空白对照组均升高(均P0.05)；加入相同浓度的顺铂, siRNA-3组细胞生存率明显高于siRNA-NC组和空白对照组(均P0.05)。流式结果显示：siRNA-3联合顺铂组细胞凋亡率明显低于顺铂组(P0.01),siRNA-3组细胞凋亡率低于对照组(P0.05)；western blot结果显示：siRNA-3组中AKT蛋白表达水平较对照组升高(P0.01),顺铂组中AKT蛋白表达水平较对照组降低(P0.01),siRNA-3联合顺铂组中AKT蛋白表达水平较顺铂组升高(P0.05)。CCK-8法检测显示：哌立福辛在48 h对ES2细胞的最小有作用剂量为4μM,以金氏公式计算结果提示4 μM哌立福辛+40μM顺铂联合用药协同抑制细胞增殖作用最强;与哌立福辛或顺铂单独作用组相比,DAPI染色发现联合用药组镜下可见大量的细胞核碎裂；联合用药能够协同增加细胞凋亡率；协同抑制细胞有丝分裂；联合用药能够明显增加细胞中Caspase-3 (cleaved)蛋白的表达,降低PARP(cleaved)蛋白的表达(P0.01)。研究结论：采用siRNA干扰片段沉默卵巢透明细胞癌ES2细胞中ARID1A基因表达后能够降低ES2细胞对顺铂的敏感性,减少细胞的凋亡；推测其机制可能涉及蛋白激酶B(AKT)信号通路的激活。AKT抑制剂哌立福辛联合顺铂能够协同抑制卵巢透明细胞癌ES2细胞的增殖并诱导肿瘤细胞发生凋亡：其促凋亡机制可能是通过上调Caspase-3 (cleaved)及下调PARP(cleaved)蛋白的表达实现的。
[Abstract]:The aim of this study was to design and synthesize siRNA targeting human ARID1A gene in vitro , transfected human ovarian clear cell carcinoma ES2 cells , screened the effective fragment of down - regulated ARID1A expression , studied the effect of ARID1A gene silencing on cisplatin sensitivity , and discussed possible molecular pathways change ;
In vitro proliferation inhibition of ES2 cells was investigated by the combination of protein kinase B inhibitor and cisplatin in vitro . The synergistic effect and possible mechanism of apoptosis were discussed .
RT - PCR and western blot were used to detect the silencing of ARID1A gene from mRNA and protein levels respectively . The best fragment ( i.e . siRNA - 3 ) was screened . After 24 hours of transfection of ES2 cells with siRNA - 3 fragment , different concentrations of cisplatin were added . After 48 h incubation , median inhibitory concentration ( IC50 ) was determined by CCK - 8 method .
After transfection , the survival rate of each group was measured after 48 h incubation with the same concentration of cisplatin ( 50 渭M ) .
The apoptosis rate of ES2 cells in each experimental group was detected by flow cytometry . The expression level of protein kinase B was detected in ES2 cells of each experimental group by western blot .
Combined with cisplatin ( DDP ) treatment for 48 h with the minimum effective dose of piperacillin , the most obvious combination dose was screened by using the gold formula . The apoptosis of ES2 cells was treated with DAPI staining and flow cytometry .
The mitotic index of the cells was detected by Gigi staining .
The expression level of Caspase - 3 was detected by western blot . Results : After transfection of ES2 cells with ARID1A - specific siRNA - 3 fragment , CCK - 8 method showed that the IC50 of siRNA - 3 group was higher than that of siRNA - NC group and blank control group ( all P0.05 ) .
The survival rate of siRNA - 3 group was significantly higher than that in the control group ( P0.05 ) . The results showed that the apoptosis rate of siRNA - 3 combined with cisplatin group was significantly lower than that in the cisplatin group ( P0.01 ) , and the apoptosis rate of siRNA - 3 group was lower than that of control group ( P0.05 ) .
The results of western blot showed that the expression level in the siRNA - 3 group was higher than that in the control group ( P0.01 ) . The expression level in the cisplatin group was lower than that in the control group ( P0.01 ) .
the combination therapy can increase the cell apoptosis rate ;
Synergistic inhibition of cell mitosis ;
Combined use of siRNA could significantly increase the expression of Caspase - 3 protein in the cells and decrease the expression of parp ( P0.01 ) . Conclusion : The sensitivity of ES2 cells to cis - platinum can be reduced and the apoptosis of the cells can be reduced after the expression of ARID1A gene in ES2 cells of ovarian clear cell carcinoma is silenced by siRNA interference fragments .
It is speculated that the mechanism may involve the activation of the signaling pathway of protein kinase B ( PKC ) , which can synergistically inhibit the proliferation of ovarian clear cell carcinoma ES2 cells and induce apoptosis of tumor cells : its pro - apoptotic mechanism may be achieved by up - regulation of Caspase - 3 ( Caspase - 3 ) and down - regulation of the expression of parp - protein .

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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